This application relates to an improved method for determination of the sequence of DNA and to an apparatus for practicing such a method.
Sequencing of nucleic acids using the chain termination method involves the general steps of combining the target nucleic acid polymer to be sequenced with a sequencing primer which hybridizes with the target nucleic acid polymer; extending the sequencing primer in the presence of normal nucleotide (A, C, G, and T) and a chain-terminating nucleotide, such as a dideoxynucleotide, which prevents further extension of the primer once incorporated; and analyzing the product for the length of the extended fragments obtained. Analysis of fragments may be done by electrophoresis, for example on a polyacrylamide gel.
Although this type of analysis was originally performed using radiolabeled fragments which were detected by autoradiography after separation, modem automated DNA sequencers generally are designed for use with sequencing fragments having a fluorescent label. The fluorescently labeled fragments are detected in real time as they migrate past a detector.
U.S. Pat. No. 5,171,534 which is incorporated herein by reference describes a variation of this basic sequencing procedure in which four different fluorescent labels are employed, one for each sequencing reaction. The fragments developed in the A, G, C and T sequencing reactions are then recombined and introduced together onto a separation matrix. A system of optical filters is used to individually detect the fluorophores as they pass the detector. This allows the throughput of a sequencing apparatus to be increased by a factor of four, since the four sequencing reaction which were previously run in four separate lanes or capillaries can now be run in one. However, it also requires the development of four compatible labels, and potentially complicated optical systems to allow discriminatory excitation and/or detection of these labels.
International Patent Publication No. WO97/40184 describes a method for increasing throughput of a DNA sequencer by altering the manner in which labels are applied to the various samples. In this method, each sample is first divided into four aliquots which are combined with four sequencing reaction mixtures. Each sequencing reaction mixture contains a polymerase enzyme, a primer for hybridizing with the target nucleic acid, nucleoside feedstocks and a different dideoxynucleotide. This results in the formation of an A-mixture, a G-mixture, a T-mixture and a C-mixture for each sample containing product oligonucleotide fragments of varying lengths. The product oligonucleotide fragments are labeled with fluorescent tags, and these tags will generally be the same for all four sequencing reactions for a sample. However, the fluorescent tags used for each sample are distinguishable one from the other on the basis of their excitation or emission spectra. Next, the A-mixtures for each sample are combined to form a combined A mixture, the G-mixtures are combined to form a combined G-mixture and so on for all four mixtures. The combined mixtures are loaded onto a separation matrix at separate loading sites and an electric field is applied to cause the product oligonucleotide fragments to migrate within the separation matrix. The separated product oligonucleotide fragments having the different fluorescent tags are detected as they migrate within the separation matrix.
Nelson et al., xe2x80x9cDNA Sequencing of four bases in three lanesxe2x80x9d Nucleic Acids Res. 20: 1345-1348 (1992) and Nelson et al., xe2x80x9cSequencing two DNA Templates in five channels by digital compressionxe2x80x9d Proc. Natl Acad. Sci. (USA)90:1647-1651 (1993) discuss the use of numerical coding approaches to reduce the number of lanes of a gel necessary to determine a DNA sequence. Reductions to 3 lanes or 2.5 lanes are achieved, respectively, by mixing combinations of dideoxy terminators in the various reactions.
The present invention provides an alternative approach to improving the throughput of a sequencing apparatus. Furthermore, the present invention makes it possible to explicitly determine the positions of all four bases in a single lane of a separation medium while utilizing only three fluorescent labels. In accordance with the invention, the sequence of a target DNA molecule is determined by preparing four chain termination reaction mixtures, one for each base type, as follows:
(a) a first set of chain-termination fragments indicative of the positions of a first type of base, wherein the fragments in the first set of fragments are labeled with a first fluorescent label;
(b) a second set of chain-termination fragments indicative of the positions of a second type of base, different from the first type of base, wherein the fragments in the second set of fragments are labeled with a second fluorescent label different from the first fluorescent label;
(c) a third set of chain-termination fragments indicative of the positions of a third type of base, different from the first and second types of bases, wherein the fragments in the third set of fragments are labeled with a third fluorescent label, different from the first and second fluorescent labels or with at least two labels, including at least one fluorescent label selected from among the first, second and third fluorescent labels; and
(d) a fourth set of chain-termination fragments indicative of the positions of a fourth type of base, different from the first, second and third types of bases, wherein the fragments in the fourth set of fragments are labeled differently from the third set of chain-termination fragments and with at least two different species of labels including at least one fluorescent label selected from among the first, second and third fluorescent labels. Thus, a total of only three fluorescent labels are required to label the DNA sequencing fragments.
The first, second, third and fourth sets of chain-termination fragments are loaded onto the same lane of an electrophoresis separation medium and separated in an electric field. The separated fragments are detected in real-time as they migrate in the electrophoresis separation medium by irradiating the separated fragments with an excitation beam and collecting light emitted by the fluorescent labels in three optical channels. The signals from three optical channels are evaluated to determine a DNA sequence for the target species.
A further aspect of the present invention is an apparatus adapted for performing sequencing in accordance with this methodology. Such an apparatus has a separation medium holder, and a power source and electrodes for applying an electric field to separation medium. The apparatus also has one or more excitation sources and optical systems appropriate for directing the excitation light to detection sites in the separation medium; and detectors for detecting light in three optical channels, one for detection of each of the three labels. The time-domain signals from the three optical channels are analyzed using a computer processing system, which can be located in the same housing as the balance of the electrophoresis apparatus or in a separate apparatus which receives the signal from the electrophoresis apparatus.